LEU3 gene sequence of S. cerevisiae and use in regulation of amino acid synthesis

ABSTRACT

An analysis of LEU3, a leucine-specific regulatory locus encoding a factor for control of RNA levels of a group of leucine-specific genes, is provided. 
     DNA sequence analysis of a clone of LEU3 shows that it contains an open reading frame of 886 amino acids. There are three regions of particular interest: a cluster of acidic amino acids that are located in the C-terminal half of the coding region, a region with a repeated cysteine motif, and a region of partial homology with MATalpha2. A LEU3-dependent DNA binding activity is demonstrated to interact with homologous portions of the 5&#39;-region of LEU1 and LEU2. 
     The sequencing of the LEU3 gene, and the identification of the key sequences within the gene, provide a means for producing the protein in deficient yeast strains and non-yeast strains, for modifying the gene in the key regions to thereby alter DNA binding with LEU1, LEU2, LEU4 and other genes for proteins and expression of LEU1, LEU2, LEU4, and LEU3 gene products, and for use within other nucleotide sequences for expression in different protein frameworks. In one application, synthesis of leucine, valine, and isoleucine, as well as the expression of the enzymes specific to leucine biosynthesis, alpha-isopropyl malate synthase, IPM isomerase, and beta-IPM dehydrogenase, may be induced or enhanced.

The United States government has rights in this invention by virtue ofNational Institute of Health grant No. GM15539.

BACKGROUND OF THE INVENTION

In bacteria, operons for amino acid biosynthetic enzymes are controlledspecifically by their respective end-product amino acids. In yeast,however, many of the genes encoding amino acid biosynthetic enzymes aresubject to regulation by the general amino acid control system. Thisregulatory network consists of a hierarchy of proteins whose function isto modulate the levels of a number of amino acids in the cell.Starvation for any one of these amino acids leads to derepression of allof the genes subject to general control.

Previous studies of enzymes involved in the biosynthesis of leucine inS. cerevisiae revealed a combination of both general and specific aminoacid regulation. LEU4, which encodes the first enzyme in the pathway,alpha-isopropyl malate synthase, abbrieviated alpha-IPM synthase, issubject to regulation by both the general amino acid control system andspecifically by leucine through feedback inhibition. LEU1 and LEU2,which encode the second and third enzymes in the pathway, respectively,are only under specific amino acid control. Expression of both genes isrepressed by elevated levels of leucine. This sensitivity to a specificamino acid seems to distinguish these genes from most others that areassociated with amino acid biosynthesis in yeast.

The leucine-specific control of LEU1 and LEU2 is thought to be indirect.Expression of these two genes appear to be a function of the level ofalpha-IPM, the product of the first enzyme in the pathway. This is basedon the following results: (1) the levels of the LEU1 and LEU2 geneproducts are sharply decreased in a strain that lacks a functionalsynthase; (2) leu1 and leu2 mutants, which are expected to accumulateintermediates in the pathway (such as alpha-IPM), exhibit increasedlevels of beta-IPM dehydrogenase and alpha-IPM isomerase, the productsof the LEU2 and LEU1 genes, respectively; and (3) a strain that containsa feedback resistant alpha-IPM synthase and produces high levels ofalpha-IPM, also has increased levels of the LEU1 and LEU2 gene products,as reported by Baichwal et. al., in Current Genetics 7,369-377(1983) andBrisco et al in Genetics 115,91-99(1987). Because alpha-IPM synthase isfeedback inhibited by leucine, the levels of alpha-IPM are directlyrelated to the levels of leucine in the cell. Accordingly, if alpha-IPMfunctions as an inducer, it could be responsible for the indirect,leucine-specific control of LEU1 and LEU2.

A single genetic locus having the potential for being a factor in theregulation of LEU1 and LEU2 was uncovered by analysis of mutantsdefective in leucine biosynthesis. This mutant allele, designated leu3,is a leaky leucine auxotroph which produces low levels of both the LEU1and LEU2 gene products and is described by Kohlhaw in "Regulation ofLeucine Biosynthesis in lower Eukaryotes", Amino Acid Biosynthesis andGenetic Regulation, L. H. Herrmann and R. L. Somerville, eds.,285-299(Addison-Wesley 1983). One hypothesis is that the LEU3 gene productfunctions as a positive activator of LEU1 and LEU2, in conjunction withalpha-IPM as an inducer.

The nucleotide sequences of LEU1 and LEU2 have been established and ithas been shown that levels of the respective mRNAs are sensitive tointracellular leucine concentrations (Andreadis et al., Cell 31, 319-325(1982); Hsu and Schimmel, J. Biol. Chem. 259,3714-3719(1984); Andreadiset al., J. Biol. Chem 259, 8059-8062 (1984)). This work also establisheda section of partial nucleotide sequence homology between the 5'-regionsof the two genes.

To date, the sequence of LEU3 has not been known, nor its exact role inregulation of amino acid synthesis, specifically, the interaction of thegene product of LEU3 with LEU1 and LEU2.

It is therefore an object of the present invention to provide a furthercharacterization of the role of LEU3 in the leucine-specific regulationof the LEU1 and LEU2 genes.

It is a further object of the present invention to provide the sequenceof the LEU3 gene and to define the roles of portions of the sequenceinvolved in DNA binding and gene regulation.

It is a still further object of the present invention to provide a meansfor inducing or regulating protein synthesis using the product of theLEU3 gene.

SUMMARY OF THE INVENTION

Although the majority of genes for amino acid biosynthesis which havebeen examined are under general amino acid control, LEU1 and LEU2respond specifically to leucine. Provided is an analysis of LEU3, aleucine-specific regulatory locus encoding a factor for control of RNAlevels of a group of leucine-specific genes. LEU3 is shown to benecessary for expression of wild type levels of LEU1- and LEU2-specificRNAs. Further, the levels of LEU4-specific transcripts are affected byLEU3.

DNA sequence analysis of a clone of LEU3 shows that it contains an openreading frame of 886 amino acids. A striking feature of the predictedLEU3 protein is a cluster of acidic amino acids (19 out of 20) that arelocated in the C-terminal half of the coding region. The protein alsohas a repeated cysteine motif and a region of partial homology toMATalpha2 and the homeo box domain.

Whole cell extracts containing a LEU3-dependent DNA binding activity aredemonstrated to interact with the 5'-region of LEU1 and LEU2.Subdivision of the LEU2 5'-region establishes that the LEU3-dependentbinding activity interacts only with the segment of LEU2 which ishomologous with LEU1.

The LEU3-dependent DNA binding activity is useful in the induction orregulation of protein synthesis including not only expression of LEU1,LEU2, and LEU4, but also any gene wherein the sequences homologous tothe 5' regions of LEU1, LEU2, LEU4, and ILV2 are incorporated into the5' region of the gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a deletion analysis of the LEU3 locus. Various fragments ofthe LEU3 complementing plasmids pPF701 and pPF702 were cloned into yeastshuttle vectors containing 2 microns DNA and the URA3 gene. The insertsizes of pPF701 and pPF702 are 10.7 and 11.2 kbp respectively. PlasmidpPF711 was constructed by deleting a 5.5 kbp BstE11 fragment frompPF702, the result of which is to fuse a distal 1.5 kbp insert fragmentto the remainder of the insert. The open rectangles represent insertDNA; vector sequences are represented by the thin lines. The hatchedarea corresponds to the leu3 open reading frame. Complementation wasmeasured in strain PDY139-11B as the ability of transformants to grow onminimal media without leucine or with 0.6 mM Tfl.

FIG. 2 is the nucleotide and deduced amino acid sequence of the LEU3gene. The region from +20 to +2790 was sequenced on both strands. Thenumbering of the DNA sequence, in parentheses, is from the start oftranslation. The amino acids are numbered from the proposed initialmethionine. The region of acidic amino acids is underlined. The regioncontaining the repeated cysteine residues is underscored with dots. Thedashed lines underscore the region of partial homology with MATalpha2and the homeo box domain. The major transcription initiation sites, asdetermined by S1 mapping, are indicated by asterisks.

FIG. 3 is a comparison of potential leucine regulatory sequences foundupstream of a number of yeast genes involved in branched chain aminoacid biosynthesis.

FIG. 4 is a diagram of the LEU3-dependent protein binding to theupstream regions of LEU1 and LEU2.

(A) Organization of the LEU2 upstream region. Numbering is relative tothe start of translation. ORF indicates the leucine-rich open readingframe. The 5'-end of the major LEU2 transcript is indicated by the wavyarrow. The putative leucine-specific upstream activating site (UAS_(L))is indicated by the hatched rectangle. The sizes of fragments B (170 bp)and C (114 bp) are inconsistent with the size of fragment A (279 bp) dueto the filling in of 5 base "overhangs" that were generated in thecleavage of fragment A to yield fragments B and C.

(B) Analysis of LEU3-dependent protein binding to the LEU2 upstreamregion using the gel retardation assay. Extracts were prepared fromstrains PDY102-1A (LEU3) and PDY139-11B (leu3). ³² P-labelled fragment Awas used as substrate. F indicates free DNA. Major complexes areindicated by arrows.

(C) Analysis as in FIG. 4B except for the replacement of fragment A withfragment B or C as indicated. The amount of extract per reaction was 40micrograms.

(D) Analysis as in FIG. 4C except that a 282 bp Xba1-Cla1 fragment ofthe LEU1upstream region was used as substrate.

DETAILED DESCRIPTION OF THE INVENTION

The sequence and properties of the LEU3 gene, as depicted in FIGS. 1-4,were obtained as follows:

Isolation and Initial Characterization of LEU3-Containing Plasmids:

In order to select a genomic clone of LEU3 by plasmid transformation,strain PDY139-11B was constructed. The relevant genotype of this strainis leu3-781 ura3-52 LEU4^(r) -103. The product of the LEU4 allele, whichis no longer subject to feedback inhibition by leucine, confers adominant resistance to the toxic analog trifluoroleucine. Because of thelow levels of the LEU1 and LEU2 gene products in leu3 strains,PDY139-11B has a leucine auxotrophy which renders the cells sensitive totrifluoroleucine. The introduction of LEU3-containing plasmids intoPDY139-11B imparts both leucine prototrophy and trifluoroleucineresistance.

PDY139-11B was transformed with DNA from a yeast genomic library. Thislibrary was constructed by ligating yeast chromosomal DNA, which hadbeen partially digested with Sau3A., into the BamHI site of themulti-copy shuttle vector YEp24 (Carlson and Botstein, Cell28,145-154(1982)). The vector also carries the URA3 gene as a selectablemarker. Approximately 9000 Ura⁺ transformants were screened for eitherleucine prototrophy or resistance to trifluororleucine. From this screen10 independent Leu⁺ /Tfl^(r) transformants were obtained.

Four of the transformants were chosen for further study. Each was grownunder non-selective conditions and was monitored for loss of theplasmid-borne URA3 marker and of the Leu⁺ /Tfl^(r) phenotypes. Each ofthese transformants co-segregated the Ura⁺ and Leu⁺ /Tfl^(r) phenotypes.As an additional test that the Leu⁺ /Tfl^(r) phenotypes are plasmiddependent, total DNA was prepared from each transformant, the plasmidswere isolated by transformation of E.coli and were then reassayed bytransforming strain PDY139-11B. All of the resulting Ura⁺ transformantswere also Leu⁺ /Tfl^(r).

Dissection and Further Analysis of the LEU3 Locus:

Restriction mapping of plasmid isolates identified two distinct typesthat are designated pPF701 and pPF702. These plasmids, which each conferLeu⁺ /Tfl^(r) on PDY139-11B, have in common a 3.8 kbp yeast genomic DNAsegment. Based on this observation, a number of deletion plasmids wereconstructed to further define the LEU3 locus (FIG. 1). This analysisestablishes that the smallest region common to the plasmids which fullycomplements the leu3 allele is a 3.4 kbp segment that extends from theBstE11 site to the Nhel site.

Whether cloned DNA which complements LEU3 can direct integration to theLEU3 locus in the yeast chromosome was then tested. This was achieved bycloning a 4.6 kbp Hindlll fragment (same as the LEU3 segment in plasmidpPF741 (FIG. 1)) into the yeast integrating vector Ylp5 (Struhl et al.,Proc.Nat'l.Acad.Sci.U.S.A. 76,1035-1039(1979)). This plasmid containsURA3 as a selectable marker but has no means for autonomous DNAreplication in yeast. To direct integration to the LEU3 locus, theconstructed plasmid was linearized by cleavage at the unique BstE11 sitewithin the insert. This DNA was then transformed into a ura3-52 his5strain designated F23. Stable Ura⁺ transformants were crossed toPDY139-11B and the resulting diploid was sporulated and subjected totetrad analysis.

The results, summarized in Table 1, show that the integrated URA3 geneis tightly linked to the wild type LEU3 allele. However, theheterozygous H1S5 marker in the diploid segregates randomly with respectto LEU3 and URA3. These observations suggest that the 4.6 kbp Hind111fragment contains sequences that direct integration to the LEU3 locus.

                  TABLE I                                                         ______________________________________                                        Cloned DNA Directs Integration at the                                         LEU3 Locus                                                                                 Number of Spores                                                 Phenotyes    Displaying Phenotype                                             ______________________________________                                        Leu.sup.+ Ura.sup.+                                                                        37                                                               Leu.sup.+ Ura.sup.-                                                                        0                                                                Leu.sup.- Ura.sup.+                                                                        0                                                                Leu.sup.- Ura.sup.-                                                                        41                                                               Leu.sup.+ His.sup.+                                                                        19                                                               Leu.sup.+ His.sup.-                                                                        18                                                               Leu.sup.- His.sup.+                                                                        21                                                               Leu.sup.- His.sup.-                                                                        20                                                               ______________________________________                                    

The LEU3 Locus has been mapped to chromosome X11. This was accomplishedby hybridizing LEU3-specific probes to a blot of intact yeastchromosomes that were separated by orthogonal-field-alternation gelelectrophoresis, according to the method of G. F. Carle and M. V. Olson,Nuc.Acids Res. 12,5647-5664 (1984). The result is in agreement with themap position of LEU3 recently reported by P. R. G. Brisco et al.,Genetics 115,91-99 (1987). Brisco et al., reported the cloning of LEU3along with preliminary genetic studies, but not the sequence. He alsoshowed that the presence of a LEU3 plasmid clone boosts the productionof LEU1 and LEU2 proteins in defective yeast cells, although not themeans by which it did so.

Effect of LEU3 on the Levels of LEU1- and LEU2-Specific RNAs:

The Northern blot hybridization method was used to detect LEU1- andLEU2-specific RNAs in isogenic strains that differ only at the LEU3locus. These strains, PDY102-1A (LEU3) and PDY139-11B (leu3), containthe LEU4^(r) allele. As a result, neither strain is feedback inhibitedby leucine and therefore is unimpeded in the synthesis of alpha-IPM. Thelatter is proposed to be a co-factor in the induction of synthesis ofthe LEU1 and LEU2 gene products.

The denatured RNA was resolved on an agarose/formaldehyde gel,transferred to nitrocellulose and probed with the appropriate ³²P-labeled fragment.

The results show that the level of LEU2-specific RNA is dependent onLEU3. This was the first time that it has been shown that the LEU3 genehas a direct effect on RNA levels. Introduction of the LEU3 plasmidpPF711 into a leu3 strain returns the LEU2-specific RNA to levelsobserved in the LEU3 strain. It is of interest that LEU3 on a multi-copyplasmid does not increase the level of LEU2 RNA substantially above thatobserved in PDY102-1A, which contains a single chromosomal copy of LEU3,indicating that a co-factor for LEU3 function, such as alpha-IPM, islimiting.

Similar hybridization experiments show that the level of LEU1 RNA isalso dependent on LEU3.

An RNA blot was probed with DNA from the HIS3 and LEU2 genes. HIS3 isknown to be controlled by the general amino acid control system. Theresult shows that HIS3 RNA levels are not sensitive to the nature of theLEU3 allele. This is expected if the effects of LEU3 are distinct fromthose of the general amino acid control system.

Effect of LEU3 on the Levels of LEU4-Specific

RNA:

The effect of LEU3 on the transcription of LEU4 was also examined.Regulation of LEU4 expression by leucine (as opposed to feedbackregulation acting on the gene product) has not previously been observed.The recently determined sequence of LEU4 contains a few regions ofpartial homology to those 5'-regions of LEU1 and LEU2 that are thoughtto be important for regulation by leucine (FIG. 3).

The results show that levels of LEU4 RNA are sensitive to the product ofthe LEU3 gene, although the effect is not as great as is observed withLEU1 and LEU2 RNA levels. The limited effect of LEU3 on LEU4 RNA may berelated to the somewhat weaker homology of LEU4 upstream sequences withthose of LEU1 and LEU2 or perhaps to the position of the sequencesrelative to the start of transcription.

The Cloned LEU3 Gene Restores Leucine

Sensitivity to LEU1 and LEU2:

LEU1 and LEU2 gene product levels are believed to be controlled throughleucine-dependent alteration in the amount of alpha-IPM. Synthesis ofthis intermediate is catalyzed by the LEU4 gene product. To determinewhether the cloned LEU3 allele confers leucine-sensitive synthesis ofLEU1 and LEU2 RNA to the leu3 strain, the leu3 allele was transferred toa LEU4 Tfl^(s) background. This strain (designated PFY400-2C), with thewild-type LEU4 allele, is subject to feedback inhibition andleucine-dependent modulation of the levels of alpha-IPM. It wastransformed with plasmid pPF711 (FIG. 1). RNA was extracted from thistransformed strain after it was grown in the absence or presence of 2 mMleucine. RNA blots were prepared and hybridized to LEU1-, LEU2- andURA3-specific probes.

In RNA isolated from cells grown in the presence of 2 mM leucine, thelevel of LEU2-specific RNA is decreased significantly. An identicalpattern of regulation by leucine is seen with LEU1-specific transcripts.These results indicate that the cloned LEU3 gene confersleucine-sensitivity to the expression of both LEU1 and LEU2 RNA.

Sequence of the LEU3 Locus:

A 3 kbp region between the BstE11 and Nhel sites of the LEU3 locus wassequenced by the dideoxy method of Sanger et al.,Proc.Nat'l.Acad.Sci.U.S.A. 74, 5463-5467 (1977). Both strands of the DNAwere sequenced independently. A single large open reading frame of 886codons was found (FIG. 2). Initiation of translation at the proposed AUGwould result in the synthesis of a protein with a calculated molecularweight of 100,127 D.

The 5'-ends of the LEU3 transcripts were determined using the S1 mappingtechnique of Berk and Sharp, Cell 12,721-732(1977). Four major mRNA5'-ends are located between bp -116 and -94. In addition, a few minortranscription initiation sites are located between bp -94 and -45. Inall cases, the first ATG downstream from the 5'-end of the mRNA is theproposed start of translation. There do not appear to be sequencesupstream of the mapped transcription initiation sites with stronghomology to the TATA sequence thought to be important in eukaryoticpromoters. Sequences that correspond to one of the proposed yeasttermination sites are located 50 bp downstream from the UAA stop codon.

There are three distinct features of the LEU3 coding sequence. The moststriking feature of the LEU3 coding region is a run of 19 out of 20acidic amino acids, spanning codons 678 to 697 (FIG. 2), which consistmostly of glutamic acid residues. The LEU3 coding region also containssequences homologous to two proposed DNA binding domains, a lysine richregion with a repeated cysteine motif, which is highly conserved in afew other yeast proteins thought to be involved in gene regulation,located in the amino terminal region of LEU3 (codons 37 to 67, FIG. 2)and a short stretch of amino acids with partial homology to a portion ofthe homeo box domain which is conserved in the MATalpha2 and MATa1 genes(codons 349 to 361, FIG. 2). While the homology in the latter case isnot extensive, the conserved amino acids include those found by Porterand Smith, in Nature 320,766-768(1986), to be essential for both haploidand diploid functions in MATalpha2.

LEU3-Dependent DNA Binding Activity:

The possibility that a LEU3-dependent product is a regulatory protein byvirtue of interactions with the 5'-regions of LEU1 and LEU2 was thenexamined. Extracts from a LEU3 strain were compared to those from leu3cells to test for a LEU3-dependent DNA binding activity that recognizesthe upstream regions of LEU1 and LEU2. The gel retardation technique,which can detect protein-DNA complexes because the mobility of a DNAfragment in a polyacrylamide gel is shifted upon protein binding, asdescribed by Fried and Crothers, Nuc.Acids Res. 9,6505-6525(1981) andGarner and Revzin, Nuc.Acids Res. 9,3047-3060(1981), was used. Theextent of the shift depends on the number of bound proteins and theirmolecular mass.

Cell extracts for binding experiments were prepared from strainsPDY102-1A (LEU3) and PDY139-11B(leu3). A Hinc11 fragment of 279 bp thatextends from -405 to -126 (FIG. 4A, fragment A) was used to examinebinding to the LEU2 upstream region. The fragments are numbered withrespect to ⁺ 1 as the start of the coding region. This fragment containsthe region of homology with LEU1. After combining the end-labeled DNAwith the extracts, the resulting complexes were separated on a 4%non-denaturing polyacrylamide gel. The experiments were done in thepresence of a 2000-fold excess of sonicated salmon sperm DNA toeliminate non-specific complexes.

Both extracts give rise to two distinct complexes, although the gelmigration patterns of these complexes are different. The amount of themajor complex detected with the LEU3 extracts is much greater than thatof the corresponding complex seen when extracts from leu3 cells areused. However, a smaller (faster migrating) complex is seen in the leu3lane, suggesting that a binding protein present in the LEU3 extracts isabsent or decreased in size. In addition, a large complex seen withincreased amounts of the LEU3 extracts is not detected with the leu3extracts. The intensity of all of the bands observed is dependent on theamount of protein extract added to the binding reaction.

FIG. 4 shows the results of experiments which define more precisely thelocation of the 5'-region of LEU2 which interacts with theLEU3-dependent DNA binding activity. The 279 bp Hinc11 fragment wascleaved with Hga1 to generate two fragments that are designated B (170bp) and C (114 bp). Fragment C contains the region of LEU2 with partialhomology with the 5'-region of LEU1. Only fragment C forms complexes hatare dependent on LEU3. The intensity of the bands observed with fragmentC is decreased by the addition of unlabeled fragment A.

Experiments were also conducted with a DNA fragment from the 5'-regionof LEU1. This 282 bp fragment extends from -309 to -27. The pattern ofLEU3-dependent complex formation is similar to that observed with theLEU2 DNA fragments.

The sequencing of the LEU3 gene, the identification of the key sequenceswithin the gene, and the characterization of a LEU3 dependent bindingactivity provide a means for producing the LEU3 gene product indeficient yeast strains and non-yeast strains, for inducing andregulating the expression of the gene products of LEU1, LEU2, LEU4, ILV2and other leucine-specific proteins through binding of the homologoussequences with the LEU3 dependent binding activity, and for use incontrolling expression of other proteins by incorporating the homologoussequences into the 5' region of the gene encoding the protein to beexpressed and then interacting the gene with the LEU3 dependent bindingactivity. One application that follows is the induction or enhancementof the synthesis of leucine, valine, and isoleucine, as well as theexpression of the enzymes specific to their biosynthesis,alpha-isopropyl malate synthase, IPM isomerase, and beta-IPMdehydrogenase.

Materials and Methods:

Strains and Genetic Methods

The following yeast strains were used: PDY139-11B (alpha LEU4^(r) -103leu3-781 ura3-52), Peter Drain, Massachusetts Institute of Technology,Cambridge, Mass.; PDY102-1A (a LEU4^(r) -103 ura3-52), Peter Drain, MIT;F23 (a his5 ura-52), Peter Drain, MIT; PFY400-2C (alpha his5 ura3-52leu3-781), a segregant derived from the cross F23 X PDY139-11B. Yeastgrowth media were prepared and general yeast methods performed asdescribed by Sherman et al., Methods in Yeast Genetics (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. 1979). Yeast weretransformed by the method of A. Hinnen et al., Proc.Natl.Acad.Sci.U.S.A.75,1929-1933 (1978) or H. Ito et al., J.Bacteriol. 41,459-472(1983).

The bacterial strains HB101 and JM101 were used for maintaining pBR322-and M13-derived plasmids, respectively. Bacterial methods were asdescribed R. W. Davis et al., Advanced Bacterial Genetics (Cold SpringHarbor Laboratory,Cold Spring Harbor, N.Y. 1980).

Hybridization Procedures

Yeast RNA for Northern hybridization was prepared as described byCarlson and Botstein, Cell 28,145-154(1982). RNA's were resolved onagarose/formaldehyde gels as described by Maniatis et al., MolecularCloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982)except that the gels were run in 10 mM NaPO₄ buffer (pH 7.0) and blotteddirectly after washing in sterile double distilled H₂ O.

Yeast DNA for chromosome blots was prepared as described by Schwartz andCantor (1984). Orthogonal-field-alteration gels were run and blotted tonitrocellulose as described by Carle and Olson Nuc.Acids Res.12,5647-5664(1984).

Pre-hybridization and hybridization conditions were as follows: 50%formamide, 5X SSPE, 5X Denhardts solution and 0.5% SDS at 37° C. DNAprobes (10⁷ cpm) labeled by nick translation according to the method ofManiatis et al.,Molecular Cloning 1982) Were incubated with the filterfor 12 hours. The final wash for all blots was 0.1X SSPE 0.1% SDS at 37°C. (55° C. for chromosome blots).

Plasmid Constructions

The details of the plasmid constructions are as follows: pPF711,deletion of 5.5 kbp BstE11 fragment from plasmid pPF701; pPF712,deletion of Sal1 fragment from pPF702 which extends leftward from theinsert Sal1 site to a Sal1 site in adjacent vector sequences; pPF715,deletion of a Sal1 fragment from pPF701 which extends rightward from theinsert Sal1 site to a Sal1 site in the adjacent vector sequences;pPF741, insertion of the 4.6 kbp Hind111 fragment of pPF701 into theHind11 site of plasmid Ylp5 (Struhl et al., Proc.Natl.Acad.Sci.U.S.A.76,1035-1039(1979)), followed by the insertion of the EcoRl fragment ofYEp24 (Botstein et al., 1979) containing the 2 micron replicationfunctions into the unique vector EcoR1 site; pPF750, deletion of an Nhelfragment from pPF702 which extends from the insert Nhe1 site rightwardto an Nhe1 site in the adjacent vector sequences; pPF751, plasmid pPF750was digested with both Nhe1 and Avrl1 (which have complementary"overhangs") and re-ligated to delete the region between the two sites.

DNA Sequence Analysis

Large fragments of plasmids pPF701 and pPF702 were cloned into the M13vectors mp18 and mp19. The DNA inserts were sequenced by the chaintermination method of Sanger et al., Proc.Natl.Acad.Sci.U.S.A.74,5463-5467(1977) as modified for [alpha-³⁵ S]dATP by Biggin et al.,Proc.Natl.Acad.Sci.USA 80,3963-3965(1983). DNA sequence information wasinitially obtained using commercially available sequencing primers(obtained from New England Biolabs). Extended stretches of DNA weresequenced by synthesizing additional sequencing primers with aMicrosyn-1450A automated DNA synthesizer (Systec, Inc., Minneapolis,Minn.)

Gel Retardation Assays

Yeast extracts were prepared and assays performed as described byArcangioli and Lescure, EMBO J.4,2627-2633(1985). Complexes wereseparated on 4% polyacrylamide gels run with 0.5X TBE buffer. The DNAfragments used as substrates were purified from 4% polyacrylamide gelsby electroelution and labeled with ³² P using polynucleotide kinase orKlenow enzyme (large fragment, DNA polymerase 1).

Although the present invention has been described with reference tospecific embodiments, variations and modifications of the disclosedsequences and protein or polypeptides regulating the synthesis ofleucine-specific amino acids will be obvious to those skilled in theart. For example, the substitution of one or two nucleotides for one ofthe listed nucleotides in the sequences regulating binding of theprotein would be obvious to those of reasonable skill in moleculargenetics and protein synthesis. It is intended that such modificationsand variations will fall within the scope of the appended claims.

We claim:
 1. A factor for control of RNA levels of a group ofleucine-specific genes comprising the amino acid sequence:

    D E E E E E D E D E E G E E E E E E E E,

wherein the factor is prepared from a cell extract.
 2. A factor forcontrol of RNA levels of a group of leucine-specific genes comprisingthe amino acid sequence:

    C V E C R Q Q K S K C D A H E R A P E P C T K C A K K N V P C,

wherein the factor is prepared from a cell extract.
 3. A factor forcontrol of RNA levels of a group of leucine-specific genes comprisingthe amino acid sequence:

    N S E L V N E Q I R T W I,

wherein the factor is prepared from a cell extract.
 4. The factor ofclaim 1 wherein the factor is expressed from cDNA.
 5. The factor ofclaim 2 wherein the factor is expressed from cDNA.
 6. The factor ofclaim 3 wherein the factor is expressed from cDNA.
 7. A polypeptideproduced by recombinant technololgy substantially comprising:

    ______________________________________                                        R E Q L N H A N L D S S V S T D I K D T E A V N E P L P I                     G R N A E H P A N Q P P L S I T Q M Q E N T L P A T Q A N                     S S L L E T Y P I V Q S N P V T T T I K E S P N S I M A G W                   D N W E S D M V W R D V D I L M N E F A F N P K V.                            ______________________________________                                    


8. A purified protein having the 886 amino acid sequence shown belowproduced by recombinant technology ##STR1##